Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/36—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Actinomyces; from Streptomyces (G)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/10—Tetrapeptides
  • C07K5/1002—Tetrapeptides with the first amino acid being neutral
  • C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
  • C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• a secretory protein has been generally known to be translated as a prepeptide or prepropeptide and thereafter to be formed into a mature protein. That is to say, in general, it has been known that it is translated as a prepeptide or prepropeptide, then the signal peptide ("a pre-part") is cleaved, thereby it is converted into a mature peptide or propeptide by further cleaving of the pro-part with a protease.
• a signal sequence refers to the sequence which is located at the N-terminal of a secretory protein precursor and which is not present in a naturally occurring mature protein
• a signal peptide refers to the peptide which is cleaved from such a protein precursor.
• the signal peptide which is used in the present invention is the signal peptide of a secretory protein from the host, Coryneform bacterium, and preferably it is the signal peptide of a cell surface protein from a Coryneform bacterium.
• Cell surface proteins include PS1 and PS2 derived from C. glutamicum (JP-Kokai No. 6-502548), and SlpA derived from C. Ammoniagenes (JP-Kokai No. 10-108675).
• the amino acid sequence of PS1 is shown in SEQ ID NO:2, the amino acid sequence of PS2 in SEQ ID NO:1 and the amino acid sequence of SIpA is shown in SEQ ID NO:3. Additionally, it is reported that DNase from a coryneform bacterium also had a signal peptide, as described in US Patent No. 4965197, which can be also used in the present invention.
• a mature transglutaminase having the same structure as that of a naturally occurring form can be efficiently produced by introducing similarly both SAMP45 gene and svPEP gene into a coryneform bacterium to which preprotransglutaminase gene has been introduced, and by allowing the bacterium to secreto-produce protransglutaminase and SAM45 as well as svPEP extracellularly or at the surface of the cells.
• the DNA probe was then generated by conducting the reaction using amplified DNA fragment of about 1.0 kb with [ ⁇ - 32 P]dCTP and Random Primer DNA Labeling Kit Ver. 2( Takarashuzo Co. Ltd.) according the protocol attached to the Kit. It was confirmed that the transglutaminase gene was present in the fragment of about 4 kb excised with restriction enzyme Sac I by Southern blot hybridization using the generated probe and the chromosomal DNA of S. mobaraense IFO13819 according to the conventional method, as described in Molecular Cloning 2nd edition [J. Sambrook, E. F. Fritsch and T. Maniatis, Cold Spring Harbor Laboratory Press, p9. 31 (1989)].
• SEQ ID NO: 12 and SEQ ID NO: 17 were synthesized based on the sequence of the transglutaminase gene determined in Example 1(1), and the region of the preprotransglutaminase gene was amplified using PCR method from pUITG obtained in Example 1(1).
• SEQ ID NOs: 12 and 17 PCR primer
• the amplified fragment of about 1.8 kb was detected by agarose electrophoresis. This fragment was recovered from the agarose gel using EASYTRAP Ver. 2 ( Takarashuzo Co. Ltd.) and inserted into Smal site of pVC7 described in JP-Kokai No. 9-070291 to obtain pVKSPTG1. The nucleotide sequence of the inserted fragment was determined according to the aforementioned method and it was confirmed that the expected fusion gene was constructed.
• the fragment of the heterologously fused prepro-transglutaminase gene which was ligated to the signal sequence of the cell surface protein SlpA of C. ammoniagenes and the 5'-upstream region comprising the promoter region of PS2 gene, was amplified by performing cross-over PCR with SEQ ID NO: 34 and SEQ ID NO: 33 using the mixture comprising 1 ⁇ l of PCR solution of the amplified region encoding the gene for the protransglutaminase derived from C. cinnamoneum IFO12852 and 1 ⁇ l of PCR solution of the amplified region comprising 5'-upstream region containing the promoter region of the PS2 gene and the region containing the signal sequence of the cell surface protein SlpA of C. ammoniagenes, as the templates.
• pUKSPTG2 having the sequence 5'-CTCTAGAG-3' wherein 5'-terminal was phosphorylated was then inserted and re-cyclized to construct pUKSPTG2.
• the fused preprotransglutaminase gene of about 1.8 kb (the protransglutaminase gene was derived from S. cinnamoneum IFO12852) was excised by digesting pUKSPTG2 with Xbal and was recovered using agarose electrophoresis. These fragments were inserted into Xbal site of pPK4 described previously to construct pPKSPTG2.
• glutamicum ATCC13869 harboring pPKSPTG2 or pPKSPTG3 was then cultured respectively in MMTG liquid culture medium (60 g of glucose, 0.4 g of magnesium sulfate heptahydrate, 30 g of ammonium sulfate, 1 g of potassium dihydrogenphosphate, 0.01 g of ferrous sulfate heptahydrate, 0.01 g of manganese(II) sulfate pentahydrate, 450 ⁇ g of thiamine hydrochloride, 450 ⁇ g of biotin, 0.15 g of DL-methionine, 50 g of calcium carbonate per liter of distilled water, adjusted to pH 7.5) containing 25 mg/l of kanamycin at 30°C for 3 days.
• MMTG liquid culture medium 60 g of glucose, 0.4 g of magnesium sulfate heptahydrate, 30 g of ammonium sulfate, 1 g of potassium dihydrogenphosphate,
• the primer shown in SEQ ID NO:45 comprises the sequence encoding the N-terminal amino acids of svPEP in order to construct the fusion gene fused to the svPEP having the pro-structure part.
• SEQ ID NOs:38 and 45 PCR primer
• the cells were removed by centrifugation and 10 ⁇ l of the supernatant of the culture was subjected to SDS-PAGE.
• the commercially available hEGF PEPRO TECHEC LTD
• CBB Coomassie Brilliant Blue
• Escherichia coli AJ12570 transformed with the plasmid pHS4 was deposited in National Institute of Bioscience and Human-Technology, Agency f Industrial Science and Technology, Ministry of International Trade and Industry (Now, Independent Administrative Agency, National Institute of Advance Industrial Science and Technology, Tsukuba Central 6, 1-1, Higashi 1-Chome Tsukuba-shi, Ibaraki-ken, 305-8566 Japan) on October 11, 1990 as FERM BP-3523.
• SEQ ID NO:56 to SEQ ID NO:59 PCR primer
• the strains grown overnight on the CM2S agar medium comprising 25 ⁇ g/l of kanamycin at 30°C were inoculated into large tubes containing 4ml of MMTG medium (Glucose 60g/L, MgSO 4 ⁇ 7H 2 O 1 g/L, MnSO 4 ⁇ 4H 2 O 1g/L, FeSO 4 ⁇ 7H 2 O 1g/L, (NH 4 ) 2 SO 4 30g/L, KH 2 PO 4 1.5g/L, VB1 - HCl 450 ⁇ g/L, Biotin 450 ⁇ g/L, DL-Met 0.15g/L, pH7.5) supplemented with 5% CaCO 3 and 25 ⁇ g/ml kanamycin, for 3 days at 30°C.
• MMTG medium Glucose 60g/L, MgSO 4 ⁇ 7H 2 O 1 g/L, MnSO 4 ⁇ 4H 2 O 1g/L, FeSO 4 ⁇ 7H 2 O 1g/

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